Pyrophosphate Gel Electrophoresis Research Articles

Heterogeneity of α-cardiac myosin heavy chains in a small marsupial, Antechinus flavipes, and the effect of hypothyroidism on its ventricular myosins

The effect of drug-induced hypothyroidism on ventricular myosin gene expression was explored in a small marsupial, Antechinus flavipes. Pyrophosphate gel electrophoresis, SDS-PAGE and western blotting were used to analyse changes in native myosin isoforms and myosin heavy chains (MyHCs) in response to hypothyroidism. In some animals, five instead of the normal three native myosin components were found: V(1a), V(1b),V(1c), V(2) and V(3), in order of decreasing mobility. In western blots, V(1a), V(1b), and V(1c) reacted with anti-alpha-MyHC antibody, but not with anti-beta-MyHC, whereas V(2) and V(3) reacted with anti-beta-MyHC antibody. SDS-PAGE of the unusual ventricular myosins revealed three MyHC isoforms, two of which bound anti-alpha-MyHC antibody while the third bound anti-beta-MyHC antibody. We conclude that V(1a), V(1b), V(1c) are triplets arising from the dimerization of two distinct alpha-MyHC isoforms. Hypothyroidism, verified by metabolic studies, decreased alpha-MyHC content significantly (t-test, P < 0.001) from 91.6 +/- 5.9% (SEM, n = 4) in control animals to 67.2 +/- 5.7% (SEM, n = 4) in hypothyroid animals, with a concomitant increase in beta-MyHC content. We conclude that in adult marsupials, ventricular myosins are also responsive to changes in the thyroid state as found in eutherians, and suggest that evolution of the molecular mechanisms underlying this thyroid responsiveness predate the divergence of marsupials and eutherians.

Left ventricular regional variations in myosin isoform shift in Dahl salt-sensitive hypertensive rats.

To evaluate the effects of chronic pressure overload on different parts of the left ventricle (LV), we examined a myosin isoform shift from V1 to V3 as a biochemical marker of LV hypertrophy in Dahl salt-sensitive (DS) rats. Six-week-old DS rats were fed an 8% (high salt, HS; n = 24) or a 0.3% (low salt, LS; n = 12) NaCl diet. After 2 or 4 weeks, the hearts were dissected and the LVs were separated into four parts (the base and mid-portion of the interventricular septum (IVS), and the base and mid-portion of the LV free wall) for isomyosin analysis. The myosin isoform shift was analyzed by pyrophosphate gel electrophoresis. Both blood pressure and LV/body weight ratio were clearly increased in the HS group. The myosin isoform shift from V1 to V3, which was measured as a decrease in the percentage of V1 isomyosin, was demonstrated only in the base of LV, with significant predominance in the IVS at 2 weeks and in all four parts at 4 weeks in the HS group. In the LS group, a myosin isoform shift was demonstrated only in the basal portion of the LV at 4 weeks. We concluded that, in rats with salt-induced hypertension, the myosin isoform shift from V1 to V3 starts at the base of the LV, and particularly at the base of the IVS, and then spreads across the entire LV. These results suggest that pressure overload from hypertension may be strongest at the base of the IVS, and that LV hypertrophy may originate at the IVS base.

Altered Expression of Cardiac Myosin Isozymes Associated with the Malignant Hyperthermia Genotype in Swine

Anesthetic-induced malignant hyperthermia (MH) in humans and pigs is associated with dramatic alterations in cardiac function. However, it remains controversial as to whether MH-associated cardiac symptoms represent a primary difference of myocardium or a secondary alteration consequent to increases in the hyperthermic stress. Here the authors describe changes in myosin isoform expression in the hearts of MH-susceptible pigs with and without prior exposure to halothane. One group of pigs was diagnosed as MH susceptible by halothane challenge and Hal-1843 nucleotide examination. To determine if there is an effect of halothane exposure, another group of pigs was diagnosed by simple MH genotyping without exposure to halothane. After diagnosis and genotyping, animals with and without exposure to halothane were killed to study cardiac myosin isozyme distributions, cardiac myofibrillar adenosine triphosphatase (ATPase) activity, and the steepness of the Ca2+-ATPase activity relation in the hearts of normal and susceptible pigs. The altered myosin isozyme expression was analyzed by pyrophosphate gel electrophoresis. Malignant hyperthermia-susceptible animals with the prior halothane challenge showed an increased V1 myosin (-44%) expression, increased myofibrillar ATPase activity (-25%) and increased steepness of the Ca2+-ATPase activity relation. Without exposure to halothane, no change of myofibrillar ATPase activity was found in the hearts of different genotyped pigs, but there was a small increase in expression of V1 myosin (-5%) in the mutant (TT). The potential modulation of V1 myosin expression occurs in the hearts of MH-susceptible pigs. The added stress by halothane challenge would further cause a V3 --> V1 shift, which may be attributed to the long-term effects of hyperthermic stress.

Jaw-closing muscles of kangaroos express alpha-cardiac myosin heavy chain.

The masseter muscle of eutherian grazing mammals typically express beta or slow myosin heavy chain (MyHC). Myosins in the masseter of 4 species of kangaroos and a slow limb muscle of one of them were compared with their cardiac myosin by pyrophosphate and sodium dodecyl sulphate (SDS) gel electrophoresis, immunoblotting and immunohistochemistry. It was found that ventricular muscle contains three isoforms homologous to V1 (alpha-MyHC homodimer), V2 (heterodimer) and V3 (beta-MyHC homodimer) of eutherian cardiac muscle, and that the masseter contained V1, with traces of V2 and V3, in great contrast to eutherian ruminants, which express only V3. A polyclonal antibody (anti-KJM) was raised in rabbits against red kangaroo masseter myosin. After cross-absorption against limb muscle myofibrils, anti-KJM specifically reacted in Westerns with MyHCs from masseter but not limb muscles, and immunohistochemically with masseter, but not limb muscle fibers. In pyrophosphate Western blots, anti-KJM reacted with V1 but not with V3. However, a monoclonal antibody specific for eutherian slow myosin stained all kangaroo slow muscle fibers but only weakly stained scattered fibers in the masseter. The SDS-PAGE revealed that light chain composition of masseter and ventricular myosins is identical, but isoforms of both light chains of kangaroo limb slow myosin were observed. These results confirm that kangaroo jaw muscle express alpha-MyHC rather than beta-MyHC. The difference in MyHC gene expression between marsupial and eutherian grazers may be related to the fact that kangaroos are not ruminants, and have only a single chance to comminute food into fine particles, hence the need for the greater speed and power of muscle contraction associated with V1 containing muscle fibers.

Age-related difference in cardiac adaptation to chronic hypertension in rats, with and without nifedipine treatment.

Three myosin isozymes, V1 (alphaalpha MHC = Myosin Heavy Chain gene), V2 (alphabeta MHC) and V3 (betaalpha MHC) that are identified in the cardiac ventricles of most mammals have been shown to shift to a V3 predominance pattern during cardiac growth and in response to left ventricular pressure overload, and to V1 predominance following anti hypertensive treatment. This study examined whether long-term hypertension impairs the ability of the adult heart to restructure myosin isozyme proportions. Using pyrophosphate gel electrophoresis, we studied proportions of cardiac myosin isozymes (V1 and V3) in young (16 weeks) and adult (36 weeks) spontaneously hypertensive rats (SHR), and following 12 weeks of nifedipine (N) treatment in age-matched SHR rats (SHR-N). The values of V1 and V3 myosin isozymes were derived by adding half of the value of V2 to each isozyme proportion. The V3 proportion in the young SHR control (SHR-C) group (49%) was 34% higher (p < 0.05) than in the young Wistar Kyoto control (WKY-C) group (37%). However, the proportion was similarly high, though not statistically significant, in both the adult SHRC (73%) and WKY-C (71%) groups. The proportion in the young SHR-N group (29%) was 41% lower (p < 0.05) than in the young SHR-C group (49%), and the proportion in the adult SHR-N group (47%) was 34% lower (p < 0.05) than in the adult SHR-C group (73%). The ratio of left ventricular weight to body weight (LVW/BW), which determines left ventricular hypertrophy (LVH), was higher in both young and adult SHR-C (26%, p < 0.05, and 42%, p < 0.05, respectively) than in WKY-C groups. The mean LVW/BW was 27% (p < 0.05) greater in adult than in young SHR-C rats. The LVW/BW in both age groups of treated SHR-N was similar to that in age matched WKY-C rats. Our study showed that a rise in the V3 level occurs in young hypertensive rats, but no rise occurs in the V3 level in adult hypertensive rats. High blood pressure seems to contribute to the high V3 level in young hypertensive rats, but in adult hypertensive rats, high blood pressure does not accentuate the V3 rise already acquired due to the aging process. Nifedipine treatment in both young and adult hypertensive rats prevented the V3 rise due to hypertension and to the aging process. This effect of nifedipine seems to be through its antihypertensive action.

Myocardial adaptive changes and damage in ischemic heart disease.

Changes in two of the elements of myocardial subcellular organelles relating to cardiac energetics, ventricular myosin isozymes and mitochondrial DNA mutations, were examined using left ventricular tissue samples obtained at autopsy from patients with ischemic heart disease. Myosin isozymes were examined in tissues from nine patients with ischemic heart disease and 12 control patients with cancer but no heart disease. Extracted myosin was separated by pyrophosphate gel electrophoresis. The relative concentration of each component was determined by densitometry. Mitochondrial DNA mutations were evaluated in tissues from ten patients with myocardial infarction and 11 control patients with cancer but no heart disease. DNA was extracted and mitochondrial DNA mutations were detected by the polymerase chain reaction. Two bands were revealed by pyrophosphate gel electrophoresis. These contained VM-A, which exhibited faster electrophoretic mobility and was present in lower concentrations, and VM-B, which had a lower mobility and a higher concentration, respectively. SDS polyacrylamide gel electrophoresis showed that these two components contained the heavy chain and light chains 1 and 2 of myosin. VM-A concentrations tended to be higher in patients with ischemic heart disease than in controls. A 7.4-kb deletion was detected between the D-loop and the ATPase 6 genes of mitochondrial DNA from the myocardium of 6 out of 10 patients with myocardial infarction. The relative amounts of the two myosin isozymes could be altered by ischemic heart disease, although the functional significance of these components is unclear. The changes in the two myosin isozymes might be an adaptive change to disordered energy metabolism, but this change was small. The myocardial mitochondrial DNA deletions in patients with myocardial infarction were thought to result from ischemic damage.

Myosin isoforms and their light chains from the ventricular muscle of the urodelan amphibian Pleurodeles waltlii: Comparison with myosin from skeletal muscles

Myosin extracted from ventricular muscle of the urodelan amphibian Pleurodeles waltlii was analyzed in comparison with myosin extracted from skeletal muscles by native, one-dimensional SDS gel electrophoresis and two-dimensional gel electrophoresis. Two myosin isoforms were detected in ventricular muscle using pyrophosphate gel electrophoresis. These isomyosins contained two types of light chain subunits, LClv and LC2v. Two-dimensional gel electrophoresis showed that LClv comigrated with the slow light chain LCls, whereas LC2v was characterized by a specific mobility, distinct from LC2s and LC2f. Diaphragm muscle was characterized by the coexistence of larval and adult myosin isoforms.

Depressed unloaded sarcomere shortening velocity in acute murine coxsackievirus myocarditis: myocardial remodelling in the absence of necrosis or hypertrophy.

We used right ventricular papillary muscles to study cellular dysfunction in acute murine coxsackievirus myocarditis. We measured unloaded sarcomere shortening velocity (V0) with laser diffraction (HeNe, lambda = 623.8 nm) 7 days after coxsackievirus infection (M) (n = 7) and after infection + monoclonal antibodies to eliminate T cells (T) (n = 4) and in normals (N) (n = 8). A servomotor rapidly shortened a muscle until slack early in contraction and V0 was measured at the onset of zero force. V0 in N was 4.14 +/- 0.84 microns.s-1 at Sl = 2.08 +/- 0.09 microns, 1.70 +/- 0.33 microns.s-1 at 2.06 +/- 0.08 microns in M and, in preliminary experiments, 4.75 +/- 0.96 microns.s-1 at 2.06 +/- 0.07 microns in T. Resting force and stiffness were normal in M. Ventriculor weights in M and T were the same as N. There was an increase in mononuclear cells in M papillary muscles, but no fibrosis or necrosis. Thus, V0 was markedly reduced in acute viral myocarditis in the absence of tissue disruption or hypertrophy, but not if T cells were absent. Pyrophosphate gel electrophoresis showed a shift from predominantly fast in N to a slow myosin isoform in M. Myosin remodelling and reduced unloaded sarcomere shortening velocity occur early in acute coxsackievirus myocarditis and are dependent on immune responses to the virus, but are not a result of histopathological changes.

Electrically evoked torque-velocity characteristics and isomyosin composition of the triceps surae in young and elderly men.

The electrically evoked isokinetic torque-velocity relationship of the triceps surae of eight elderly and four non-trained young men was examined in relation to the isomyosin composition of the soleus and the gastrocnemius muscles, determined under non-denaturing conditions using pyrophosphate gel electrophoresis. The angle specific torque-velocity properties of the triceps surae were measured using maximal percutaneous electrical stimulation at 50 Hz and a release technique. The elderly subjects generated significantly (P < 0.05) less absolute torque at all angular velocities when compared with the young subjects. When the isokinetic data were normalized to the isometric torque, the lower normalized torques generated by the elderly subjects were not statistically different from the young. The total fast isomyosin (FM) content of the soleus and gastrocnemius in the elderly subjects was 22 +/- 13 and 35+/- 18%, respectively. This compared with 29 +/- 8 (n.s) and 44 +/- 8% (n.s.) in the young subjects. When the gastrocnemius and soleus muscles were given an equal weighting and considered together to represent the whole triceps surae, the normalized torque at the fixed angular velocity of 5 rads s-1 was significantly associated with %FM (r = 0.90, P < 0.01), and the isomyosin bands %FM1 (r = 0.90, P < 0.01) and %FM2 (r = 0.93, P < 0.001) when only the elderly subjects were considered. No relationships were observed between contractile characteristics and contractile protein profile when only the young subjects were considered. This was despite the inclusion of a further two sprint and three endurance trained athletes to increase the range of contractile characteristics and differences in muscle composition.

Evidence for two myosin types in indirect insect flight muscles

Purified myosins from the asynchronous indirect flight muscles of a fleshfly ( Phormia terrae-novae) and the synchronous indirect flight muscles of the locust ( Locusta migratoria) were analyzed. In glycerol-SDS-PAGE one type of heavy chain from both flight muscle myosins had nearly identical mobility (≈204 and ≈205 kDa) the second heavy chain differed (≈202 kDa, fleshfly; ≈215 kDa, locust). Isoelectric focusing and densitometric scanning showed relative amounts of the heavy chains of 37–45% and 55–63% in either flight muscle. In pyrophosphate gel electrophoresis two types of native myosins with a molecular ratio of 1.3–1.5/1 were separated. Rotary shadowed myosin molecules from both flight muscles showed two populations with different rod lengths in nearly identical amounts.

Myocardial contractility and ventricular myosin isoenzymes as influenced by cardiac hypertrophy and its regression.

Changes in myocardial contractility and ventricular myosin isoenzymes were examined during pressure-overloaded cardiac hypertrophy in rats. Effects of regression of cardiac hypertrophy were also examined. Cardiac hypertrophy was induced by abdominal aortic constriction in 7-week-old male Wistar rats. Regression of cardiac hypertrophy was obtained by opening the aortic band. Myocardial contractility was estimated by measuring isometrically developed tension and maximum rate of tension rise (+dT/dtmax) in isolated left-ventricular papillary muscles perfused with Tyrode solution (32 degrees C, pH 7.4, bubbled with 95% O2.5% CO2, stimulation frequency: 0.2 Hz). Left-ventricular myosin isoenzymes were separated by pyrophosphate gel electrophoresis and the isoenzyme pattern was determined by densitometry. Isometrically developed tension (T) in hypertrophic myocardium remained unchanged, but +/-dT/dtmax decreased as compared with hearts of normal rats. Decreased +/-dT/dtmax recovered near to the level in normal rats by regression of cardiac hypertrophy. Left-ventricular myosin isoenzyme pattern shifted towards VM-3 in hypertrophied myocardium and shifted again toward VM-1 by regression of cardiac hypertrophy. In conclusion, myocardial contractility and ventricular myosin isoenzymes were changed in pressure-overloaded hypertrophy in rats and these changes were reversible to a normal level by regression of cardiac hypertrophy.

Myocardial contractility and energetics in cardiac hypertrophy and its regression.

The changes in myocardial contractility and ventricular myosin isoenzymes were investigated in rats with pressure-overload cardiac hypertrophy as well as during its regression. Hypertrophic myocardium was obtained from rats with renovascular hypertension (Goldblatt rats), rats with abdominal aortic constriction (AC), and spontaneously hypertensive rats (SHR). Regression of cardiac hypertrophy was induced by lowering the blood pressure through nephrectomy on the affected side in Goldblatt rats, by opening the clip which constricted the abdominal aorta in AC rats, and by the administration of antihypertensive agents to SHR. The isometric developed tension of isolated left ventricular papillary muscles and the maximum rate of increase in the tension (dT/dtmax) were measured. Left ventricular myosin isoenzymes were separated by pyrophosphate gel electrophoresis. Isometric developed tension remained unchanged, but dT/dtmax was decreased in hypertrophic myocardium, although it recovered along with the regression of cardiac hypertrophy. The left ventricular myosin isoenzyme pattern was shifted towards V3 in hypertrophic myocardium, and shifted back again towards V1 with the regression of cardiac hypertrophy. These results indicate that relief of hemodynamic overload is one of the most important elements in the regression of cardiac hypertrophy and the associated physiological or biochemical alterations. However, other factors such as neurohumoral influences must also be taken into consideration.

Different biventricular remodelling of myosin and collagen in pulmonary hypertension.

1. To clarify the metabolism of contractile and non-contractile proteins of the ventricles during the development of right ventricular hypertrophy (RVH) and accompanying congestive heart failure (CHF) in response to a pressure overload, monocrotaline was injected subcutaneously into Sprague-Dawley (SD) rats. Myosin isoenzymes (MIE) were analysed by pyrophosphate gel electrophoresis under non-dissociating conditions. Acid-soluble collagens were analysed by an improved, noninterrupted sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tissue collagen content was also measured after the estimation of hydroxyproline concentration in tissues. 2. Monocrotaline induced RVH, but not left ventricular hypertrophy, at 2 weeks after the injection of monocrotaline, with severe RVH with CHF present at 4 weeks. In the right ventricles (RV) treated with monocrotaline, MIE shifted significantly from V1 to V3 at 2 weeks. The shift of MIE was more pronounced at 4 weeks. The proportion of type III collagen increased significantly compared with controls at 2 weeks. At 4 weeks, the proportion of types III and V collagens increased significantly compared with controls. 3. In left ventricles (LV) treated with monocrotaline, a similar but less remarkable shift of MIE was observed without remodelling of collagen types at 2 and 4 weeks. The concentration of collagen in either the RV or LV treated with monocrotaline showed no significant changes at 2 and 4 weeks compared with controls. 4. These results demonstrate a remodelling of the contractile and non-contractile proteins during the development of RVH and accompanying CHF, and provide evidence for changes in protein metabolism of the counterpart of RV (i.e. the LV). These results may provide important insights into the pathophysiology of adaptive or non-adaptive cardiac hypertrophy in response to a pressure overload.

Sliding velocity of isolated rabbit cardiac myosin correlates with isozyme distribution.

To investigate the relationship between the mechanical and biochemical properties of cardiac myosin, the sliding velocity of isolated cardiac myosin obtained from both euthyroid and hyperthyroid rabbits on actin cables was measured with an in vitro motility assay system. Ten rabbits (T) were treated with L-thyroxine to induce hyperthyroidism, and eight nontreated animals (N) were used as controls. Myosin was purified from the left ventricles of anesthetized animals. Myosin isozyme content was analyzed by the pyrophosphate gel electrophoresis method, and myosin adenosinetriphosphatase (ATPase) activity was determined on the same sample. Long well-organized actin cables of green algae, Nitellopsis, were used in the in vitro motility assay. Small latex beads were coated with purified cardiac myosin and introduced onto the Nitellopsis actin cables. Active unidirectional movement of the beads on the actin cables was observed under a photomicroscope, and the velocity was measured. The velocity was dependent on ATP concentrations, and the optimal pH for bead movement was approximately 7.0-7.5. The mean velocity was higher in T than in N (0.66 +/- 0.12 vs. 0.32 +/- 0.09 micron/s, P less than 0.01). Both Ca(2+)-activated ATPase activity and the percentage of alpha-myosin heavy chain were also higher in T than in N (0.691 +/- 0.072 vs. 0.335 +/- 0.072 microM Pi.mg-1.min-1, P less than 0.01, and 79 +/- 12 vs. 26 +/- 7%, P less than 0.01, respectively). The velocity of myosin closely correlated with both Ca(+2)-activated myosin ATPase activity (r = 0.87, P less than 0.01) and the percentage of alpha-myosin heavy chain (r = 0.87, P less than 0.01).

Effects of regression of cardiac hypertrophy on myocardial contractility and ventricular myosin isoenzymes.

The effects of regression of cardiac hypertrophy on myocardial contractility and ventricular myosin isoenzymes were investigated in rats with renovascular hypertension. Six-week-old male Wistar rats were made hypertensive by constriction of one renal artery with a silver clip. Regression of cardiac hypertrophy was induced following the lowering of blood pressure by nephrectomy on the affected side 5-6 weeks after constriction of the renal artery and was maintained for 5-6 weeks. In contrast, myocardial hypertrophy was induced by 10-11 weeks of the hypertensive state. Isometric developed tension of isolated left ventricular papillary muscles was measured, while they were being perfused with Tyrode solution. Left ventricular myosin isoenzymes were separated by pyrophosphate gel electrophoresis. The ventricular to body weight ratio of the nephrectomized group was significantly lower than that of the hypertensive group, although it was greater than that of age-matched normal control rats. There were no significant differences in the isometric developed tension among three groups, the nephrectomized, hypertensive, and normal control rats. However, dT/dtmax tended to decrease in the hypertensive rats and recovered to normal in the nephrectomized rats. The left ventricular myosin isoenzyme pattern was shifted toward VM-3 in hypertensive rats and was shifted back toward VM-1 again in nephrectomized rats.

Effects of long term treatment with an 1 adrenoceptor blocker on cardiac hypertrophy, contractility, and myosin isoenzymes in spontaneously hypertensive rats

The aim was to investigate the effects of long-term treatment with an alpha 1 blocker, bunazosin hydrochloride, on blood pressure, heart weight, myocardial contractility, and ventricular myosin isoenzyme pattern in spontaneously hypertensive rats (SHR). Bunazosin hydrochloride was given orally in a dose of 10 mg.kg-1.d-1 for 8-9 weeks. Isometric tension development was measured in isolated left ventricular papillary muscles. The left ventricular myosin isoenzyme pattern was determined by pyrophosphate gel electrophoresis. 14 male SHR (seven treated and seven untreated rats) aged 23 to 24 weeks were studied. The blood pressure of the bunazosin treated rats was approximately 14% lower than that of the untreated rats at the end of the treatment period. The ventricular weight of treated SHR was significantly lower (around 8%) than that of untreated rats, but there were no significant differences between the two groups in the mechanical data obtained from isolated left ventricular papillary muscles. The myosin isoenzyme pattern of the left ventricle was significantly shifted toward VM-1 in the bunazosin treated SHR, the VM-1 concentration in the treated group being 38% greater than in the untreated group. These results indicate that long term treatment with bunazosin hydrochloride reduces blood pressure and leads to the regression of cardiac hypertrophy in SHR. Myocardial energetics (as represented by the myosin isoenzyme pattern) were affected, but there was no influence on myocardial tension development.

Modification in the expression and localization of contractile and cytoskeletal proteins in Schwartz-Jampel syndrome

Muscle biopsies taken from 4 patients with clinical diagnosis of Schwartz-Jampel syndrome were analyzed by enzyme-histochemical immunocytochemical and biochemical techniques. In situ distribution of the different myosin heavy chain (MHC) isoforms together with that of the cytoskeletal proteins vimentin, desmin and titin was determined in type I, type IIA, type IIB and type IIC fibers. The same muscle biopsies were analyzed for their content in myosin light chains (MLC) by two-dimensional gel electrophoresis and native myosin isoforms by pyrophosphate gel electrophoresis. The opportunity to study 4 patients of different ages, all members of the same family, permitted us to reveal several interesting features in this rare and so far poorly understood muscle pathology. (i) We observed a predominance of slow (type I) fibers in the oldest patient. (ii) Two classes of small clusters of atrophic type IIC fibers were observed. The first class corresponded to fibers which coexpressed embryonic, fetal and fast, but not slow, MHC isoforms. The fibers also displayed an abnormal distribution of desmin, vimentin and titin. The second class was composed by fibers coexpressing embryonic, fetal, fast and slow, MHC isoforms. In contrast to that observed for the first class, fibers in the second class displayed a normal pattern of expression of desmin, vimentin and titin. (iii) A familial heterogeneity was observed between the 4 patients. The pathological processes involved in the evolution of this syndrome are discussed.

A novel myosin heavy chain isoform in vascular smooth muscle

Previous studies demonstrated two myosin heavy chain isoforms in vascular smooth muscles with SDS-polyacrylamide gel electrophoresis; MHC 1 (204kDa) and MHC 2 (200kDa). We report the existence of a novel myosin heavy chain isoform, MHC 3 (196kDa), which was exclusively contained in inferior vena cava. Equal amount of MHC 1 and MHC 2 was observed in aorta and pulmonary artery, respectively. However, inferior vena cava contained only MHC 3. Proteolytic artifact was refuted by immunoblotting of tissue homogenates without purification, or SDS-polyacrylamide gel electrophoresis of myosin bands isolated by pyrophosphate gel electrophoresis. Furthermore, α-chymotryptic cleavage of MHC 1, MHC 2, and MHC 3 displayed different peptide maps, indicating the primary structural difference among all three isoforms.

Metabolic and contractile protein expression in developing rat diaphragm muscle

Progressive changes in myosin isozyme expression and in energy-generating enzyme activities were followed in the diaphragm and, for comparison, in axial and appendicular muscles of rats from 18 d gestation to maturity. Native myosins were characterized by pyrophosphate gel electrophoresis. Myosin heavy-chain (MHC) isozymes were measured with ELISA using monoclonal antibodies and were localized by immunocytochemistry. RNA transcripts for the MHCs were demonstrated on Northern blots and by RNase protection assays. Quantitative activities of malate dehydrogenase (MDH), beta-hydroxyacyl CoA dehydrogenase (beta OAC), 1-phosphofructokinase (PFK), lactate dehydrogenase (LDH), creatine kinase (CK), and adenylokinase (AK) were measured in muscle homogenates and in individual fibers by fluorometric pyridine nucleotide-dependent assays. Compared to limb muscles, expression of neonatal myosin in the diaphragm is precocious. Neonatal MHC mRNA is prominent in the diaphragm at 19 d gestation, and neonatal myosin is the major MHC isoform present at birth. Slow and fast IIa MHCs are also present at birth. Transcripts for IIa MHC are detectable in the diaphragm at 21 d gestation and are upregulated at birth. Comparable signal for IIa MHC mRNA is not found in the gastrocnemius until 10 d postpartum. Adult fast IIb MHC mRNA was detected only as a faint signal at 30-40 d in the diaphragm and then disappeared. Results indicate that a separate phenotype, the IIx type, matures late in diaphragmatic development. The activities of enzymes representing all of the major energy pathways are higher in the fetal diaphragm than in the fetal hindlimb muscles. For example, beta OAC had sixfold higher activity in the diaphragm than in the extensor digitorum longus (EDL) muscle at birth, activity in the diaphragm than in the extensor digitorum longus (EDL) muscle at birth.

Changes in Contractile and Noncontractile Protein Metabolisms in Both Ventricles in Monocrotaline-Treated Rats

To clarify the metabolism of contractile and noncontractile proteins of both ventricles (BVs) during the development of right ventricular hypertrophy (RVH) induced by pressure overload, monocrotaline (M) was injected subcutaneously into Sprague-Dawley (SD) rats. Myosin isoenzymes (MIEs) were analyzed by pyrophosphate gel electrophoresis. Acid-soluble collagens were analyzed using improved noninterrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tissue collagen concentrations were also measured. M induced RVH, but not left ventricular hypertrophy, at 2 weeks, and severe RVH at 4 weeks. In right ventricles (RVs) of M-treated rats, MIE significantly shifted from V1 to V3, and the proportions of type III and V collagens increased compared to control at 2 and 4 weeks. In the left ventricles (LVs) of M-treated rats, similar but less remarkable MIE shifts were found without remodeling of collagen types at 2 and 4 weeks. Collagen concentrations of BVs treated with M did not show any significant changes compared to control at 2 and 4 weeks. Our results show remodelings of contractile and noncontractile proteins in RVs during the development of RVH, and also provide evidence for the changes in protein metabolism of the counterpart of RVs (i.e., LVs) during the development of cardiac hypertrophy.